Structure-function relationships in ribulose 1,5-bisphosphate carboxylase/oxygenase: Heterologous expression and characterization of engineered protein.

Persistent Link:
http://hdl.handle.net/10150/185095
Title:
Structure-function relationships in ribulose 1,5-bisphosphate carboxylase/oxygenase: Heterologous expression and characterization of engineered protein.
Author:
Ramage, Robert Thomas, III.
Issue Date:
1990
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco, E.C. 4.1.1.39) is required by all photosynthetic organisms. Recombinant rubisco, generated by heterologous expression of cloned genes in E. coli, was examined to determine enzyme structure/function relationships. Recombinant higher plant rubisco LSU was insoluble and nonfunctional. This was probably due to the absence of appropriate protein assembly apparatus in the heterologous host. Recombinant higher plant rubisco SSU was present in two forms, as soluble protein and as insoluble material. Recombinant Anacystis rubisco was present as soluble protein and as insoluble material. The soluble enzyme had carboxylase activity of 2.2 μmol CO₂ fixed/min/mg rubisco. The kinetically determined parameters with respect to RuBP were K(m)(RuBP) of 38 μM and V(max) of 3.5 μmol CO₂ fixed/min/mg rubisco. Insoluble SSU from either pea or Anacystis was manipulated into soluble, functional protein by a denaturation-renaturation procedure. Insoluble LSU from either spinach or Anacystis could not be manipulated into soluble protein by the denaturation-renaturation procedure. Recombinant Anacystis LSU alone, without SSU, assembled into the octameric core structure, was able to bind CABP, and could catalyze the carboxylation reaction at a rate of approximately 1% of holoenzyme. When SSU was added it assembled with LSU to form holoenzyme, stabilized the conformation of the active site region, and increased the rate of the carboxylation reaction. Mutant LSU with a 4 aa internal addition, near an active site Lys residue, was functional but carboxylase activity was reduced to 0.4 μmol CO₂ fixed/min/mg rubisco. Mutant LSU with large additions or extensions were expressed as insoluble material. Heterologous holoenzyme, composed of Anacystis LSU and the cyanobacterial-type SSU from Cyanophora or higher plant SSU from either pea or Mesembryanthemum, was enzymetically active. Heterologous holoenzymes had reduced carboxylase activity and higher K(m)(RuBP) compared to Anacystis rubisco. Enzyme activation was inhibited when non-activated heterologous holoenzyme was incubated with RuBP. This suggests that higher plant SSU plays a role in the tight binding of RuBP to unactivated enzyme. The function of a higher plant SSU specific region (HPIN) was investigated by examining chimeric SSU in chloroplast protein import assays. Precursor SSU proteins that contained HPIN were imported, processed, and assembled into holoenzyme in isolated chloroplasts. Precursor forms of chimeric SSU without HPIN were imported and processed but did not assemble with endogenous higher plant LSU. This resulted in the identification of HPIN as a putative assembly domain. Carboxylase assays of rubisco composed of Anacystis LSU and the chimeric SSU showed that the chimeric SSU were enzymatically functional.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Chemistry
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Biochemistry; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Bohnert, Hans J.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleStructure-function relationships in ribulose 1,5-bisphosphate carboxylase/oxygenase: Heterologous expression and characterization of engineered protein.en_US
dc.creatorRamage, Robert Thomas, III.en_US
dc.contributor.authorRamage, Robert Thomas, III.en_US
dc.date.issued1990en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco, E.C. 4.1.1.39) is required by all photosynthetic organisms. Recombinant rubisco, generated by heterologous expression of cloned genes in E. coli, was examined to determine enzyme structure/function relationships. Recombinant higher plant rubisco LSU was insoluble and nonfunctional. This was probably due to the absence of appropriate protein assembly apparatus in the heterologous host. Recombinant higher plant rubisco SSU was present in two forms, as soluble protein and as insoluble material. Recombinant Anacystis rubisco was present as soluble protein and as insoluble material. The soluble enzyme had carboxylase activity of 2.2 μmol CO₂ fixed/min/mg rubisco. The kinetically determined parameters with respect to RuBP were K(m)(RuBP) of 38 μM and V(max) of 3.5 μmol CO₂ fixed/min/mg rubisco. Insoluble SSU from either pea or Anacystis was manipulated into soluble, functional protein by a denaturation-renaturation procedure. Insoluble LSU from either spinach or Anacystis could not be manipulated into soluble protein by the denaturation-renaturation procedure. Recombinant Anacystis LSU alone, without SSU, assembled into the octameric core structure, was able to bind CABP, and could catalyze the carboxylation reaction at a rate of approximately 1% of holoenzyme. When SSU was added it assembled with LSU to form holoenzyme, stabilized the conformation of the active site region, and increased the rate of the carboxylation reaction. Mutant LSU with a 4 aa internal addition, near an active site Lys residue, was functional but carboxylase activity was reduced to 0.4 μmol CO₂ fixed/min/mg rubisco. Mutant LSU with large additions or extensions were expressed as insoluble material. Heterologous holoenzyme, composed of Anacystis LSU and the cyanobacterial-type SSU from Cyanophora or higher plant SSU from either pea or Mesembryanthemum, was enzymetically active. Heterologous holoenzymes had reduced carboxylase activity and higher K(m)(RuBP) compared to Anacystis rubisco. Enzyme activation was inhibited when non-activated heterologous holoenzyme was incubated with RuBP. This suggests that higher plant SSU plays a role in the tight binding of RuBP to unactivated enzyme. The function of a higher plant SSU specific region (HPIN) was investigated by examining chimeric SSU in chloroplast protein import assays. Precursor SSU proteins that contained HPIN were imported, processed, and assembled into holoenzyme in isolated chloroplasts. Precursor forms of chimeric SSU without HPIN were imported and processed but did not assemble with endogenous higher plant LSU. This resulted in the identification of HPIN as a putative assembly domain. Carboxylase assays of rubisco composed of Anacystis LSU and the chimeric SSU showed that the chimeric SSU were enzymatically functional.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectChemistryen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorBohnert, Hans J.en_US
dc.contributor.committeememberBourque, Don P.en_US
dc.contributor.committeememberDieckmann, Carol L.en_US
dc.contributor.committeememberJensen, Richard G.en_US
dc.contributor.committeememberMatsuda, Kaoruen_US
dc.identifier.proquest9028167en_US
dc.identifier.oclc708387766en_US
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